The present invention relates generally to miniaturized liquid phase sample processing and analysis. More particularly, the invention relates to a miniaturized planar sample preparation and analysis device with an on-chip miniature electrophoresis ion analyzer and conductivity detection means. The device, including both ion analyzer and detection means are manufactured using a variety of means suitable for microfabrication of substrate materials such as, but not limited to ablation, molding and embossing.
Ion analysis is used in a wide variety of applications, including environmental applications such as water and soil analysis, food industry applications such as wine and beer analysis and in the nuclear power industry.
Currently, there are two main techniques that are used for ion analysis. The more dominant technique is ion chromatography (xe2x80x9cICxe2x80x9d) in combination with conductivity detection. In this technique, ion exchange columns are used to separate the components of a sample and conductivity measurements are used for detection since may of the ions of interest do not have properties suitable for optical detection. The advantage of IC is that a large quantity of sample can be applied to the column so that a detectable amount of any trace compounds is available for analysis. However, in IC there are problems with the stability of the detection systems and with the reproducibility of ion analysis. In addition, some ions are not easily separated using IC. An additional disadvantage of ion chromatography is the cost per analysis. IC uses expensive instrumentation, expensive columns and solvents and requires generally more sample handling.
More recently, capillary electrophoresis (xe2x80x9cCExe2x80x9d) has been used for the same applications as ion chromatography. One advantage of CE is that it separates components of a sample based on a different principle and thus is a suitable complementary technique. However, indirect UV detection is typically used in combination with CE to detect ion analytes, which does not have the sensitivity to detect trace sample components. In addition, the ability to load quantities of sample onto a CE apparatus is limited which makes CE less preferred for analysis of trace components of a sample.
Another technique that has been used for ion analysis is isotachophoresis (xe2x80x9cITPxe2x80x9d). ITP is performed by sandwiching a sample between a leading and a terminating buffer in a column or capillary and applying an electric field across the leading and terminating buffer reservoirs. The leading buffer is chosen to contain an anion or cation of greater mobility than the anions or cations, respectively, present in the sample. The terminating buffer is selected to contains ions of lesser mobility than those present in the sample. Upon application of an electric field, xe2x80x9csample stacking,xe2x80x9d i.e., the arrangement in distinct bands of sample substances in order of decreasing mobility, occurs based on the choice of electrolytes. Sample stacking makes it possible to detect trace amounts of a component in a sample. The length of the band for a particular component of the sample depends not on the concentration of the component but on its quantity.
Once separation is complete, all buffer and solute ions migrate at the same velocity. The separation velocity (xcexdISO), which is the same for all zones, is given by xcexdISO =xcexcLEL=xcexcSES=xcexcTET, in which xcexc is the mobility, E is the electric field strength and subscripts 1, s and t related to leading ion, sample ion and terminating ion. A different electric field develops in each zone because different ions have different mobilities. The highest electric field appears where the lowest mobility ions are present, i.e., the terminating buffer.
The concentration of a solute in its isotachophoretic zone is determined by the composition and concentration of the leading electrolyte. The concentration of the solute is given by CA=CL[xcexcA/(xcexcA+xcexcC)][(xcexcL+xcexcC)/xcexcL]. Since the mobility of each ion is constant under the defined conditions, the above equation can be written as CA=CLxc3x97k, in which k is a constant. Thus, the sample ion concentration is directly proportional to the leading ion concentration.
The boundary layer between two isotachophoretic zones is self-sharpening, leading to very high resolution. This self-sharpening effect is derived from the different field strengths in the various zones. For example, if an ion of type A diffuses into the trailing zone of ion of type B, it would be in an environment of lower electric field strength than its own zone. The type A ion""s velocity would be decreased from the value xcexd=xcexcAEA to the value xcexd=xcexcAEB, so it would be retarded relative to the A/B zone boundary. Similarly, if ion B diffuses into the A zone, the field strength is higher, so its velocity would be increased until it is overtake by the A/B zone boundary.
Because each component zone and each of buffer zones have different field strengths, electroosmotic flow in each region will be different region. If electroosmotic flow counter to ITP flow is so high that ITP flow is significantly impeded, the analytes will not separate as desired. To overcome this problem, the viscosity of the buffers could be increased. An alternate solution to the problem that has been used has been to place a membrane designed to stop bulk flow at each end of the ITP column or capillary.
In one technique known as column-coupling ITP, a pre-separation step is used to allow large-volume injection and sample enrichment followed by analytical separation and detection. A commonly used mode of detection in ITP is conductivity detection. Conductivity detection is not only a very sensitive means by which to detect ionic sample components it also provides information on the type of ion detected. However, disadvantages of using conductivity detection concerns the stability of the electrodes that are placed in contact with the liquid buffers and samples. In addition, analysis by ITP involves a complex buffer selection procedure and an understanding of method development for a particular sample.
Silicon micro-machining has been useful in the fabrication of miniaturized liquid phase analysis systems and improvements have been made to overcome the inherent shortcomings of this technique. For example, U.S. Pat. No. 5,500,071 to Kaltenback et al., U.S. Pat. No. RE 36,350 to Swedberg et al. and U.S. Pat. No. 6,033,628 to Kaltenback et al., disclose the use of laser ablation to form microstructures in novel polymer substrates. This permits an enhance symmetry and alignment of structures formed by component parts, enhanced separation capabilities, avoidance of problems with SiO2 chemistry, low-cost manufacturing, the formation of microstructures of any size and geometry, and the incorporation of a detection means for on-column analysis.
Accordingly, it is a primary object of the invention to provide a novel miniaturized ion analysis device.
It is yet another object of the invention to provide a method for analyzing ionic species present in a sample using the miniaturized ion analysis device.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention.
The present invention is directed to a novel miniaturized ion analysis system for liquid phase isotachophoretic sample analysis and detection. The system comprises contactless conductivity detection (xe2x80x9cCCDxe2x80x9d) means capable of detecting ionic species fluid sample.
In one embodiment of the invention, miniaturized ion analysis system for isotachophoretic sample preparation and analysis is provided. The system comprises:
a microfabricated support body having first and second substantially planar opposing surfaces wherein the support body has a microchannel microfabricated in the first planar surface;
a cover plate arranged over the first planar surface, wherein the cover plate in combination with the first microchannel forms a sample flow compartment;
an inlet port and an outlet port communicating with the sample flow compartment, wherein the inlet and outlet ports enable downstream passage of fluid from an external source through the sample flow compartment; and
a contactless conductivity detection means in electrical communication with the sample flow compartment such that the detection means is capable of detecting the presence in the sample flow compartment of a fluid containing a detectable ionic species.
In another embodiment of the invention, a miniaturized ion analysis system for isotachophoretic sample preparation and analysis is provided. The system comprises:
a microfabricated support body having first and second component halves each having substantially planar opposing interior and exterior surfaces;
a first microchannel microfabricated in the interior surface of the first support body half and a second microchannel microfabricated in the interior surface of the second support body half, wherein each of the microchannels is so arranged as to provide the mirror image of the other;
an elongate bore formed by aligning the interior surfaces of the support body halves in facing abutment with each other whereby the microchannels define a sample flow compartment;
an inlet port and an outlet port communicating with the sample flow compartment, the ports enabling the downstream passage of fluid from an external source through the sample flow compartment; and
a contactless conductivity detection means situated in electrical contact with the sample flow compartment such that the detection means is capable of detecting the presence in the sample flow compartment of a fluid containing a detectable ionic species.
In yet another embodiment of the invention, a method for analyzing ionic species present in a sample is provided. The method comprises:
providing a miniaturized ion analysis system as disclosed herein;
flushing the sample flow compartment with leading electrolyte;
introducing a sample into the sample flow compartment such that the sample is in fluidic and ionic communication with the leading electrolyte;
introducing terminating electrolyte into the sample flow compartment such that the sample is in fluidic and ionic communication with the leading electrolyte;
stacking the ionic species present in the sample by applying a current across the leading and terminating electrolytes to provide stacked ionic species; and
detecting the presence in the sample flow compartment of the stacked ionic species.
A particular advantage of the present invention is the use of processes other than silicon micromachining techniques or etching techniques to create miniaturized columns in a wide variety of polymeric, ceramic, glass, composite substrates having desirable attributes for an analysis portion of a separation system. More specifically, it is contemplated herein to provide a miniaturized ion analysis system prepared by ablating, molding or embossing component microstructures in a substrate using techniques well known in the art. In one preferred embodiment, a miniaturized ion analysis system is formed by providing two substantially planar halves having microstructures microfabricated thereon, which, when the two halves are folded upon each other, define a sample flow compartment featuring enhanced symmetry and axial alignment.
Use of microfabrication techniques, e.g., laser ablation, to form a miniaturized ion analysis system according to the present invention affords several advantages over prior etching and micromachining techniques used to form systems in silicon or silicon dioxide materials. Initially, the capability of applying rigid computerized control over such processes allows microstructure formation to be executed with great precision, thereby enabling a heightened degree of alignment in structures formed by component parts. For example, laser ablation processes avoid problems encountered with microlithographic isotropic etching techniques which may undercut masking during etching, giving rise to asymmetrical structures having curved side walls and flat bottoms.
Microfabrication, in particular, laser ablation, enables the production of microstructures with greatly reduced component size. In this regard, microstructures formed as described herein are capable of having aspect ratios several orders of magnitude higher than possible using prior etching techniques, thereby providing enhanced sample processing capabilities in such devices. For example, the use of laser-ablation processes to form microstructures in substrates such as polymers increases ease of fabrication and lowers per-unit manufacturing costs in the subject devices as compared to prior approaches such as micromachining devices in silicon. In this regard, devices formed according to the invention in low-cost substrates have the added feature of being capable of use as substantially disposable miniaturized analysis units.
Laser ablation or other microfabrication techniques used in a planar substrate allows for formation of microstructures of almost any geometry or shape. This feature not only enables the formation of complex device configurations, but further allows for integration of sample injection, sample preparation, pre- or post-separation chemical modification and a variety of detection means, in particular, a CCD means, in a miniaturized ion analysis system.
By the present invention, inherent weaknesses existing in prior approaches to liquid phase analysis device miniaturization, and problems in using silicon micromachining techniques to form miniaturized analysis devices have been addressed. Accordingly, the present invention discloses a miniaturized ion analysis system capable of liquid phase isotachophoretic sample preparation and analysis on a wide array of liquid samples.